The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent.

2008-01-01·Journal of Drug Targeting3区 · 医学

Effect of LHRH-PE40 on target cells via LHRH receptors

3区 · 医学

Article

作者: Ya-Qin Zhu ; Xin Deng ; Doreen Akkerman ; Ping Zhu ; Bing-Zhi Yu ; Guo-Li Zhang ; Ying Liu ; Sven Klussmann ; Hang Chen ; Guang-Mou Wu

OBJECTIVETo detect the effect and cytotoxicity of luteinizing hormone-releasing hormone-Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40) on target cells using LHRH receptors (LHRHR).METHODSThe affinity of LHRH-PE40 and LHRH binding to LHRHR on the membrane surface of target cells were measured by enzyme linked immunosorbent assay. Morphological observations with light microscope were used to analyze its receptor pathway, with Spiegelmer, and cytotoxicity. IC(50) values of LHRH-PE40, which caused 50% inhibition of tumor cell growth were evaluated by MTT assay. The target cells were exposed to LHRH-PE40 and its cytotoxicity was analyzed by scanning and transmission electron microscopies, agarose gel electrophoresis, and flow cytometry.RESULTSLHRH-PE40 killed target cells by LHRHR pathway. The morphological changes in these cells showed decreased cell size, cytoplasmic membrane blebbing, and chromatin condensation and margination. At a certain concentration and time point, HeLa cells were also induced to undergo programmed cell death.CONCLUSIONLHRH-PE40 induced target cells apoptosis via LHRHR.

2004-01-01·World Journal of Gastroenterology3区 · 医学

Ability of luteinizing hormone releasing hormone-Pseudomonas aeruginosaexotoxin 40 binding to LHRH receptor on human liver cancer cells

3区 · 医学

Article

作者: Zhu, Ping ; Gong, Shou-Liang ; Zhao, Gang ; Liu, Guang-Wei ; Lu, Wen-Tian ; Zhao, Hong-Guang

AIMTo explore the ability of recombinant toxin luteinizing hormone releasing hormone-Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40) and LHRH binding to LHRH receptor (LHRHR) on the membrane surface of human liver cancer HEPG cells.METHODSLHRH was labeled by using (125)I with enzymatic reaction. The affinity and receptor volume of LHRH-PE40 and LHRH binding to LHRHR on the membrane surface of human liver cancer cells were measured with radioligand receptor assay.RESULTSThe specific activity of LHRH labeled with (125)I was 2.7 x 10(4) kBq/microL, and its radiochemical purity reached to 99.2-99.7%. The binding of (125)I to LHRH was maximal for 240 min in the warm cultivation, and this binding was stabilized. The inhibiting rates of (125)I-LHRH and LHRH on the proliferation of human liver cancer HEPG cells were not significantly different. On the basis of the saturation curve of (125)I-LHRH binding to the membrane LHRHR of HEPG cells, (125)I-LHRH of 1 x 10(5) cpm was selected for radioligand receptor assay. The affinity constants (Kd) of LHRH-PE40 and LHRH binding to the membrane LHRHR of HEPG cells were 0.43+/-0.12 nmol/L and 4.86+/-1.47 nmol/L, respectively, and their receptor volumes were 0.37+/-0.15 micromol/g and 0.42+/-0.13 micromol/g, respectively. The binding of LHRH-PE40 to the membrane protein of normal liver cells was not observed.CONCLUSIONThe recombinant toxin LHRH-PE40 binding to the membrane surface of LHRHR of human liver cancer HEPG cells was very strong, while the specific binding of it to normal liver cells was not observed. The results provide an important experimental basis for the clinical application of LHRH-PE.

100 项与人性腺激素释放激素类似物-绿脓杆菌外毒素A功能片段重组蛋白相关的药物交易

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